Effect of DNA methylation on gene expression

<div><p>A. RNA from purified adult or fetal liver erythroblasts and MEFs from Cre (γ^A promoter methylated) or Mx-cre (γ^A promoter unmethylated) crossed transgenic (lines 47, 64 or 113)) mice were subjected to semi-quantitative RT-PCR on 1 or 3 µl samples to detect human β (HBB) or γ(HBG)-globin RNA. </p> <p>We measured Aprt expression to control for the amount of cDNA in the reaction.</p> <p>Genes expressed at high levels were quantitated by diluting the input sample (e.g. 1/10⁵ for Aprt in MEFs).</p> <p>In order to distinguish between the two γ genes, we took advantage of a PstI restriction site that is present in γ^A and not γ^G<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Tanimoto2" target="_blank">[51]</a>.</p> <p>A smaller induction of γ^A was observed in line 66 which is only partially unmethylated at the promoter (data not shown).</p> <p>The degree of IE excision in purified erythroblasts from induced Mx-cre carrying mice was found to be >90% by PCR analysis.</p> <p>The data for fetal erythroblasts was taken from an animal with a “methylated” γ^A gene, but we have demonstrated that it is indeed unmethylated in fetal liver (data not shown), as expected.</p> <p>B. Quantitative analysis of RNA. Levels of globin expression were obtained from dilution analysis and compared to Aprt (set to 1) in the same cells. </p> <p>Each experiment was repeated 2–3 times (coefficient of variance = 14%).</p> <p>γ/γ+β was calculated on the basis of total γ globin (γ^A+γ^G).</p> <p>Results (average of 3 experiments) for real time PCR are included for some samples.</p> <p>The degree of γ globin induction is shown for adult erythroblast cells.</p></div

Programmed methylation of the human γ^A-globin promoter

<div><p> <b>A.</b> Two IEs (yellow) bounded by loxP elements (stippled black) were inserted into the γ^A promoter region of a YAC containing the human β-globin locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Tanimoto1" target="_blank">[18]</a>, and this was used to generate transgenic mice that were then crossed with two different cre-expressing lines. </p> <p>In the first line (Cre) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Lallemand1" target="_blank">[19]</a>, cre is expressed (red) prior to implantation.</p> <p>In mice carrying this construct the IE is deleted before the wave of de novo methylation, and surrounding CpG sites thus become methylated (red circles).</p> <p>The second cre-expressing line carries interferon-inducible cre <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Kuhn1" target="_blank">[20]</a>.</p> <p>In these mice (Mx-cre), the IE remains present during implantation and protects adjacent regions from methylation.</p> <p>By treating adult animals with polyI-polyC, cre activity could be induced (red) and the IE removed, generating an unmethylated version of the transgene.</p> <p> <b>B.</b> Tail DNA from four transgenic founders (lines 47, 64, 66 and 113) was cut with AvrII with or without the methylation sensitive restriction enzyme BsrFI, and subjected to Southern blotting using a radioactive probe (green arrow). </p> <p>Line 47 was crossed with cre mice and offspring analyzed in the same way.</p> <p> <b>C.</b> The region upstream of the γ^A and γ^G coding sequences (blue triangles) was analyzed for CpG methylation using the bisulfite technique.</p> <p>To this end we generated MEFs from line 64 (IE present at time of implantation) and infected them with an adenovirus that expresses Cre activity.</p> <p>Alternatively, we derived MEFs from line 64 mice crossed with Cre (IE removed prior to implantation).</p> <p>Five CpG residues were analyzed for methylation in the γ^G promoter.</p> <p>The normal γ^A promoter also carries 5 CpGs, but one is replaced by the inserted loxP element.</p> <p>Following bisulfite treatment, PCR products were cloned and subjected to sequencing.</p> <p>Each row represents a single molecule.</p> <p>Black circles indicate a methylated CpG, whereas open circles indicate a lack of methylation.</p> <p>The numbers in the margin indicate how many clones were fully methylated.</p></div

γ promoter methylation pattern in patients with HPFH

<div><p>The γ^A and γ^G promoter regions were analyzed for CpG methylation on individual clones by the bisulfite technique.</p> <p>To this end we used Fibach culture erythroblasts derived from normal individuals, as well as heterozygote HPFH1 or β⁰ Thalassemia (IVS-II-I, G⇑A) patients.</p> <p>Black circles indicate methylated CpGs, whereas open circles indicate a lack of methylation.</p> <p>The numbers in the margin indicate how many clones were fully methylated.</p></div

IE induces undermethylation in transgenic mice

<div><p>We made transgenic embryos (12.5 dpc) using either a plasmid that contains the normal promoter of the human γ^A-globin gene (-IE), or a plasmid that contains the promoter of the human γ^A-globin gene modified to harbor a dimer of the IE bounded by two lox elements (+2IE) 40 bp upstream to the γ^A transcription start site, and tested total founder embryonic DNA for methylation at specific restriction sites by southern blot analysis. This plasmid (+2IE) was later used as a template for homologous recombination in the YAC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#s4" target="_blank">methods</a>).</p> <p>The map indicates the restriction site locations according to PubMed accession number NG_00007.</p> <p>Bold marked restriction enzymes are methylation sensitive. Total founder embryo DNA was digested with HindIII alone or together with CfoI, or alternatively with StuI/XbaI alone or together with HpaII, MspI or AvaI.</p> <p>Southern-blot analysis was performed using probes 5′ or 3′ as labeled.</p> <p>The sizes of expected bands are indicated on the autoradiogram. Data shown are representative of results obtained with several transgenic founder mice for each construct.</p> <p>From this experiment, we conclude that a double IE is capable of inducing undermethylation over a distance of at least 500 bp in each direction.</p></div

Effect of DNA methylation on promoter structure

<div><p>Mononucleosomes were prepared from either non-erythroid (<b>A</b>) or erythroid (<b>B</b>) cells taken from Cre (methylated) and Mx-cre (unmethylated) founder (64) transgene crosses and subjected to ChIP analysis using anti-Ac-H4.</p> <p>Input (I) and bound (B) DNA fractions were used for semi-quantitative PCR on 1 or 3 µl samples using primer sets specific for the γ^G or loxP-inserted γ^A promoter regions.</p> <p>For each ChIP preparation, the Acta1 gene (green) was assayed as a negative control and the Actb gene (yellow) as a positive control.</p> <p>The results are summarized in graphic form after normalizing to Acta1 (green) enrichment (set at 1.0) (coefficient of variance = 17%).</p> <p>The results for the Cre (blue) and Mx-cre (red) mice are shown.</p> <p>Results for β globin are presented for comparison.</p> <p>The data shown for non-erythroid cells was obtained using mononucleosomes from MEFs, but the graph summarizes results from 3–4 ChIP experiments on both MEFs and lymphocytes.</p> <p>(<b>C</b>) In vivo footprinting of γ promoter.</p> <p>Erythroblasts (in vivo) or purified erythroblast DNA (in vitro) from mice carrying either a methylated or unmethylated γ^A promoter were treated with DMS. LMPCR gel.</p> <p>Maxam-Gilbert lanes (AG and CT) are sequencing controls.</p> <p>The numbers are according to PubMed accession number NG_000007.</p> <p>Putative protein factor binding regions (rectangles) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000046#pone.0000046-Ikuta1" target="_blank">[26]</a>, DMS footprints, as in vivo protected (open circle) or hyper-reactive (closed circle) nucleotides, are indicated.</p> <p>The sizes of the circles represent relative differences in footprint intensities.</p> <p>DMS footprinting on spleen lymphocytes did not show any hyperreactive sites on unmethylated DNA, but did reveal some slight reactivity over the distal CCAAT box on methylated DNA.</p></div

AnkT9W increases HbA synthesis to therapeutic levels in most of the thalassemic ErPCs.

<p>(A) Net increase of Hb A % (ΔHb A, calculated by subtracting Hb A after treatment from that following treatment) is plotted against VCN (on the X axis) in the three patients groups, β0/0, β0/+ and β+/+. The total Hb A% is also represented as percentage of Hb A before (light grey) and after transduction (dark grey) with AnkT9W in the same groups (B). Figures C and D show the concomitant percentages of Hb F and α-aggregates, both of which are reduced in thalassemic ErPCs after treatment with AnkT9W. The tie bars under the X axes group different specimens from the same individual. Several specimens were harvested and transduced at different times so as to expose the variability in such experiments. “*” Sign indicates specimens treated with AnkT9Wsil2.</p

Mutations in patients.

<p>List of β-thalassemia specimens analyzed in this study.</p

HPLC profiles of thalassemic specimens analyzed after differentiation from CD34-derived cultures.

<p>The HPLC profiles of the same thalassemic β0/0 (A) and β+/+ (B) specimens were analyzed at steady state (left) and following treatment with AnkT9W (right) after differentiation starting from either CD34⁺ cells (top) or ErPCs (bottom). In the same β0/0 specimen, AnkT9W contributes to increasing Hb A synthesis from from 0% to 62% (VCN = 0.92), in the CD34⁺-derived cells or to 73% (VCN = 0.97), in the ErPCs-derived cells. In the cells from the β+/+ patient, the net Hb increase was 35% (VCN = 0.88), if CD34⁺-derived or 23% (VCN = 0.94), if ErPCs-derived.</p

Sequences of exon 1 and 2 in AnkT9W and AnkT9Wsil1/2 silent mutants.

<p>Original and corresponding mutated bases of exons 1 and 2 inside constructs are in bold letters.</p

The ankyrin insulator improves translation of the β-globin gene, and its sequence is conserved at the site of integration.

<p>(A) Expression of the chimeric Hb (expressed as a percentage) after HMBA differentiation, in MEL cell clones carrying one VCN of T9W or AnkT9W, and T9Ank2W. One way anova test, p = 0.048. (B) The β-globin mRNA (bottom panel), expressed by T9W (VCN = 0.87,1.4), AnkT9W (VCN = 0.39, 1.07, 1.47) and T9Ank2W (VCN = 1.26, 1.98) was not detected in non-erythroid cells (B-16 melanoma) but only in differentiated MEL cells (AnkT9W, VCN = 2), indicating that the transgene's transcription is tissue specific. (C) Two amplicons were amplified by PCR corresponding to the ankyrin sequence present, respectively, in the 5′ and 3′ viral LTRs. Oligonucleotides were designed to include the ankyrin element and selectively amplify fragments either from the 5′ or 3′ LTR. Only clones bearing a single AnkT9W integrant were used. The amplified fragments exhibited the expected size of 248 bp and 231 bp, indicating that the ankyrin element was faithfully replicated in both LTRs. (D) Southern blot assay of genomic DNA from single integrant clones of MEL cells carrying either T9W or AnkT9W or from MEL control. The genomic DNAs were digested with <i>Xmn</i>I restriction enzyme, which yielded the full β-globin cassette, identified by hybridization using a β-globin <i>BamH</i>I-<i>Nco</i>I probe. Clone #6 of the AnkT9W series was reloaded in higher quantity (left panel) to amplify the signal of the transgenic human β-globin gene, because it was insufficiently loaded and exhibited a weak signal on the first blot (right panel).</p